香术防感方吸嗅与滴鼻给药方式预防甲型流感H1N1模型小鼠的药效学比较研究

Comparative Study on the Pharmacodynamics of Xiangzhu Fanggan Formula(香术防感方)by Sniffing and Nasal Drops for Prevention of Influenza A H1N1 Virus Infection in Mice

目的比较香术防感方吸嗅和滴鼻两种经鼻给药方式预防甲型流感H1N1的有效性及可能作用机制。方法将56只BALB/c小鼠随机分为正常组、模型组、扎那米韦组、香囊高浓度组、香囊低浓度组、滴鼻高浓度组、滴鼻低浓度组,每组8只。香囊低、高浓度组分别给予15、30 g香术防感方香囊吸嗅,每天持续24 h;滴鼻低、高浓度组分别给予浓度为0.11、0.22 g/ml香术防感方滴鼻液滴鼻,每次20μl,每日2次;扎那米韦组给予浓度为1.025 mg/ml扎那米韦溶液滴鼻,每次20μl,每日2次;正常组、模型组滴鼻20μl生理盐水,每日2次。各组预防性干预5天。第5天给药1 h后,除正常组外,其余各组小鼠每只滴鼻35μl、50 LD50 A/PR/8/34/H1N1病毒液滴鼻,以制备甲型流感H1N1模型小鼠。于实验第5天至9天每日记录小鼠体质量及计算体质量变化率,并观察一般状态。于第9天取材,计算小鼠肺指数及肺指数抑制率;HE染色法检测肺组织病理变化,并进行肺组织病变评分;RT-qPCR法检测肺组织病毒载量;ELISA法检测上呼吸道灌洗液中分泌型免疫球蛋白A(sIgA)及血清肿瘤坏死因子α(TNF-α)、白细胞介素2(IL-2)、白细胞介素6(IL-6)、干扰素γ(IFN-γ)含量。结果实验第7、8、9天,模型组小鼠体质量变化率较同时间点正常组显著降低(P<0.05或P<0.01);实验第8、9天与模型组比较,扎那米韦组和滴鼻高浓度组小鼠体质量变化率增加(P<0.05或P<0.01)。与正常组比较,模型组小鼠肺指数、肺组织病变评分、肺组织病毒载量明显升高,血清TNF-α、IL-6、IL-2、IFN-γ含量明显升高,上呼吸道灌洗液sIgA水平明显降低(P<0.01)。与模型组比较,扎那米韦组和滴鼻高浓度组肺指数、肺组织病毒载量降低,血清IFN-γ、TNF-α、IL-2、IL-6含量降低,sIgA水平升高(P<0.05或P<0.01);除香囊低浓度组外,其余各药物干预组肺组织病变评分较模型组降低(P<0.01)。与扎那米韦组比较,香囊低浓度组和滴鼻低、高浓度组肺指数升高,各香术防感方干预组血清TNF-α、IL-2含量均升高(P<0.05或P<0.01)。与滴鼻高浓度组比较,香囊高浓度组血清TNF-α和IFN-γ含量升高,滴鼻低浓度组肺组织病毒载量升高(P<0.05或P<0.01)。扎那米韦组肺指数抑制率为80.84%,香囊高、低浓度组肺指数抑制率分别为41.61%、17.90%,滴鼻高、低浓度组肺指数抑制率分别为35.40%、25.40%。HE染色显示,模型组肺组织出现肺泡间隔增厚,肺泡塌陷,炎性细胞浸润;而各药物干预组小鼠肺组织炎症和损伤程度均有所减轻,且以扎那米韦组、滴鼻高浓度组改善最明显。结论香术防感方吸嗅和滴鼻两种给药形式均可预防甲型流感H1N1病毒感染,具有抗病毒、抗炎作用,可改善肺组织病理损伤,提高呼吸道黏膜免疫,且滴鼻给药在抑制炎症反应方面可能优于吸嗅给药,尤以滴鼻高浓度效佳。

Objective To compare the effectiveness and mechanism of Xiangzhu Fanggan Formula(香术防感方)by sniffing and nasal drops for preventing influenza A H1N1flu.Methods Fifty-six BALB/c mice were randomly divided into normal group,model group,zanamivir group,high-concentration sachet group,low-concentration sachet group,high-concentration nasal drops group,and low-concentration nasal drops group,with 8 mice in each group.In the low-and high-concentration sachet groups,15 g and 30 g of Xiangzhu Fanggan Formula sachet were used for sniffing for 24 h per day;while in the low-and high-concentration nasal drops groups,nasal drops of Xiangzhu Fanggan Formula were given at a concentration of 0.11 and 0.22 g/ml,20μl each time,twice a day;in the zanamivir group,zanamivir was given at a concentration of 1.025 mg/ml of 20μl each time,twice a day;in the normal group and the model group,nasal drops of normal saline were given at 20μl each time,twice a day.Each group was given prophylactic intervention for 5 days.On day 5,1 h after the administration of the drug,the mice in all groups except the normal group received 35μl of 50 LD50 A/PR/8/34/H1N1 viral solution as nasal drops to prepare influenza A H1N1 model mice.The body mass of the mice was recorded and the rate of change of body mass was calculated daily from day 5 to day 9 of the experiment,and the general status was observed.The mice were sampled on day 9,and the lung index and the inhibition rate of lung index were calculated;HE staining was used to detect pathological changes in lung tissues and to score lung tissue lesions;RT-qPCR was used to detect viral load in lung tissues;and ELISA was used to detect secretory immunoglobulin A(sIgA)and serum tumour necrosis factorα(TNF-α)and interleukin 2(IL-2),interleukin 6(IL-6),and interferonγ(IFN-γ)in the lavage fluid of the upper respiratory tract.Results On days 7,8 and 9 of the experiment,the rate of change in body mass of mice in the model group significantly lower than that in the normal group at the same time points(P<0.05 or P<0.01).On days 8 and 9 of the experiment,the rate of change in body mass of mice in the zanamivir group and the high-concentration nasal drops group increased when compared with the model group(P<0.05 or P<0.01).Compared with the normal group,mice in the model group had significantly higher lung index,lung tissue lesion score,lung tissue viral load,significantly higher serum TNF-α,IL-6,IL-2,IFN-γlevels,and significantly lower sIgA levels in the upper respiratory lavage fluid(P<0.01).Compared with the model group,the lung index and lung tissue viral load reduced,serum IFN-γ,TNF-α,IL-2,IL-6 levels reduced,and sIgA levels increased in the zanamivir group and the high-concentration nosal drops group(P<0.05 or P<0.01);except for low-concentration sachet group,lung tissue lesion scores of the drug intervention groups reduced compared with those of the model group(P<0.01).Compared with the zanamivir group,the lung index increased in the low-concentration sachet group and the low-and high-concentration nasal drops groups,and the serum TNF-αand IL-2 levels increased in all Xiangzhu Fanggan Formula intervention groups(P<0.05 or P<0.01).Compared with high-concentration nasal drops group,serum TNF-α and IFN-γlevels elevated in the high-concentration increased group,and lung tissue viral load elevated in the low-concentration nasal drops group(P<0.05 or P<0.01).The lung index inhibition rate was 80.84% in the zanamivir group,41.61% and 17.90% in the high-and low-concentration sachet groups,and 35.40% and 25.40% in the high-and low-concentration nasal drops groups,respectively.HE staining showed that the lung tissues of the model group showed thickening of alveolar septa,alveolar collapse,and infiltration of inflammatory cells;whereas,in each drug intervention group,the inflammation of the lung tissues of the mice and the damage reduced,and the most obvious improvement was in the zanamivir group and the high-concentration nasal drops group.Conclusion Xiangzhu Fanggan Formula by sniffing and nasal drops could both prevent influenza A H1N1 virus infection,with antiviral and anti-inflammatory effects,also could improve the pathological damage of lung tissue,and improve the immunity of respiratory mucosa.The nasal drops may be better than sachets in inhibiting inflammatory response,especially the high-concentration nasal drops showed more effective.